The MuSK-BMP pathway regulates synaptic Nav1.4 localization and muscle excitability.

The neuromuscular junction (NMJ) is the linchpin of nerve-evoked muscle contraction. Broadly considered, the function of the NMJ is to transduce a nerve action potential into a muscle fiber action potential (MFAP). Efficient information transfer requires both cholinergic signaling, responsible for the generation of endplate potentials (EPPs), and excitation, the activation of postsynaptic voltage-gated sodium channels (Nav1.4) to trigger MFAPs. In contrast to the cholinergic apparatus, the signaling pathways that organize Nav1.4 and muscle fiber excitability are poorly characterized. Muscle-specific kinase (MuSK), in addition to its Ig1 domain-dependent role as an agrin-LRP4 receptor, is also a BMP co-receptor that binds BMPs via its Ig3 domain and shapes BMP-induced signaling and transcriptional output. Here we probed the function of the MuSK-BMP pathway at the NMJ using mice lacking the MuSK Ig3 domain ('ΔIg3-MuSK'). Synapses formed normally in ΔIg3-MuSK animals, but the postsynaptic apparatus was fragmented from the first weeks of life. Anatomical denervation was not observed at any age examined. Moreover, spontaneous and nerve-evoked acetylcholine release, AChR density, and endplate currents were comparable to WT. However, trains of nerve-evoked MFAPs in ΔIg3-MuSK muscle were abnormal as revealed by increased jitter and blocking in single fiber electromyography. Further, nerve-evoked compound muscle action potentials (CMAPs), as well as twitch and tetanic muscle torque force production, were also diminished. Finally, Nav1.4 levels were reduced at ΔIg3-MuSK synapses but not at the extrajunctional sarcolemma, indicating that the observed excitability defects are the result of impaired localization of this voltage-gated ion channel at the NMJ. We propose that MuSK plays two distinct roles at the NMJ: as an agrin-LRP4 receptor necessary for establishing and maintaining cholinergic signaling, and as a BMP co-receptor required for maintaining proper Nav1.4 density, nerve-evoked muscle excitability and force production. The MuSK-BMP pathway thus emerges as a target for modulating excitability and functional innervation, which are defective in conditions such as congenital myasthenic syndromes and aging.

The Sick and the Weak: Neuropathies/Myopathies in the Critically Ill.

Critical illness polyneuropathies (CIP) and myopathies (CIM) are common complications of critical illness. Several weakness syndromes are summarized under the term intensive care unit-acquired weakness (ICUAW). We propose a classification of different ICUAW forms (CIM, CIP, sepsis-induced, steroid-denervation myopathy) and pathophysiological mechanisms from clinical and animal model data. Triggers include sepsis, mechanical ventilation, muscle unloading, steroid treatment, or denervation. Some ICUAW forms require stringent diagnostic features; CIM is marked by membrane hypoexcitability, severe atrophy, preferential myosin loss, ultrastructural alterations, and inadequate autophagy activation while myopathies in pure sepsis do not reproduce marked myosin loss. Reduced membrane excitability results from depolarization and ion channel dysfunction. Mitochondrial dysfunction contributes to energy-dependent processes. Ubiquitin proteasome and calpain activation trigger muscle proteolysis and atrophy while protein synthesis is impaired. Myosin loss is more pronounced than actin loss in CIM. Protein quality control is altered by inadequate autophagy. Ca(2+) dysregulation is present through altered Ca(2+) homeostasis. We highlight clinical hallmarks, trigger factors, and potential mechanisms from human studies and animal models that allow separation of risk factors that may trigger distinct mechanisms contributing to weakness. During critical illness, altered inflammatory (cytokines) and metabolic pathways deteriorate muscle function. ICUAW prevention/treatment is limited, e.g., tight glycemic control, delaying nutrition, and early mobilization. Future challenges include identification of primary/secondary events during the time course of critical illness, the interplay between membrane excitability, bioenergetic failure and differential proteolysis, and finding new therapeutic targets by help of tailored animal models.

An official American Thoracic Society Clinical Practice guideline: the diagnosis of intensive care unit-acquired weakness in adults

RATIONALE: Profound muscle weakness during and after critical illness is termed intensive care unit-acquired weakness (ICUAW). OBJECTIVES: To develop diagnostic recommendations for ICUAW. METHODS: A multidisciplinary expert committee generated diagnostic questions. A systematic review was performed, and recommendations were developed using the Grading, Recommendations, Assessment, Development, and Evaluation (GRADE) approach. MEASUREMENT AND MAIN RESULTS: Severe sepsis, difficult ventilator liberation, and prolonged mechanical ventilation are associated with ICUAW. Physical rehabilitation improves outcomes in heterogeneous populations of ICU patients. Because it may not be feasible to provide universal physical rehabilitation, an alternative approach is to identify patients most likely to benefit. Patients with ICUAW may be such a group. Our review identified only one case series of patients with ICUAW who received physical therapy. When compared with a case series of patients with ICUAW who did not receive structured physical therapy, evidence suggested those who receive physical rehabilitation were more frequently discharged home rather than to a rehabilitative facility, although confidence intervals included no difference. Other interventions show promise, but fewer data proving patient benefit existed, thus precluding specific comment. Additionally, prior comorbidity was insufficiently defined to determine its influence on outcome, treatment response, or patient preferences for diagnostic efforts. We recommend controlled clinical trials in patients with ICUAW that compare physical rehabilitation with usual care and further research in understanding risk and patient preferences. CONCLUSIONS: Research that identifies treatments that benefit patients with ICUAW is necessary to determine whether the benefits of diagnostic testing for ICUAW outweigh its burdens.(AU)

Sodium channel inactivation in an animal model of acute quadriplegic myopathy.

We previously demonstrated that muscle fibers become unable to fire action potentials in both patients and an animal model of acute quadriplegic myopathy (AQM). In the animal model, skeletal muscle is denervated in rats treated with high-dose corticosteroids (steroid-denervated; SD), and muscle fibers become inexcitable despite resting potentials and membrane resistances similar to those of control denervated fibers that remain excitable. We show here that unexcitability of SD fibers is due to increased inactivation of sodium channels at the resting potential of affected fibers. A hyperpolarizing shift in the voltage dependence of inactivation in combination with the depolarization of the resting potential induced by denervation results in inexcitability. Our findings suggest that paralysis in the animal model of AQM is the result of an abnormality in the voltage dependence of sodium channel inactivation.

The WldS protein protects against axonal degeneration: a model of gene therapy for peripheral neuropathy.

The WldS mouse is a spontaneous mutant that is characterized by the phenotype of delayed degeneration of transected nerves (slow Wallerian degeneration). Molecular genetic analysis identified a mutation in this animal that codes for a unique protein expressed in brain tissue of WldS mice. We asked whether the WldS phenotype, in addition to delaying axonal degeneration after axotomy, might provide neuroprotection against toxic neuropathy. In dorsal root ganglia (DRG) cultures, neurites from WldS transiently exposed to vincristine not only resisted axonal degeneration but resumed growth after withdrawal of the toxin. Neurites from wild type mice died rapidly and did not recover. To prove that the identified mutation and its protein product are responsible for the WldS phenotype, we used an adenoviral gene transfer system to deliver the WldS to rat DRG neurons. Rat neurons expressing the WldS protein were resistant to vincristine-induced axonal degeneration, confirming the functional significance of the identified gene mutation. These data provide evidence that the WldS protein can be neuroprotective against vincristine neuropathy, and possibly other disorders characterized by axonal degeneration. In addition, delivery of this gene to wild type cells can transfer the WldS phenotype, providing the possibility of "gene therapy" for peripheral neuropathy.

Painful idiopathic rigid flatfoot in children and adolescents.

Nine patients (13 feet) were identified whose primary complaints were of atraumatic-onset, chronic pain in the hindfoot exacerbated with increased activity and who had the diagnosis of idiopathic rigid flatfeet. Eight of 11 were greater than the 95th percentile in weight for their age. Exam under anesthesia showed moderate to significant improvement in hindfoot motion in 9 feet; 4 feet required fractional peroneal lengthenings. Only 5 of 11 patients have had sustained relief of pain and report unlimited activity level. Children and adolescents with painful idiopathic rigid flatfeet without known causation can have significant, persistent, disability and do not uniformly respond well to traditionally-described nonoperative Interventions.

Altered gene expression in steroid-treated denervated muscle.

In rats treated with high-dose corticosteroids, skeletal muscle that is denervated in vivo (steroid-denervated) develops electrical inexcitability similar to that seen in patients with acute quadriplegic myopathy. To determine whether changes in muscle gene transcription might underlie inexcitability of steroid-denervated muscle we performed RNase protection assays to quantitate adult (SkM1) and embryonic (SkM2) sodium channel isoforms and chloride channel (CLC-1) mRNA levels in control, denervated, steroid-innervated, and steroid-denervated skeletal muscle. While SkM1 mRNA levels were relatively unaffected by denervation or steroid treatment, SkM2 mRNA levels were increased by both. These effects were synergistic and high levels of SkM2 mRNA were expressed in denervated muscle exposed to corticosteroids. Skeletal muscle CLC-1 mRNA levels were decreased by denervation. To better understand the marked upregulation of SkM2 in steroid-denervated muscle we examined changes in myogenin and glucocorticoid receptor mRNA levels. However, changes in these mRNA levels cannot account for the upregulation of SkM2 in steroid-denervated muscle.

Disruption of Trkb-mediated signaling induces disassembly of postsynaptic receptor clusters at neuromuscular junctions.

Neurotrophins and tyrosine receptor kinase (Trk) receptors are expressed in skeletal muscle, but it is unclear what functional role Trk-mediated signaling plays during postnatal life. Full-length TrkB (trkB.FL) as well as truncated TrkB (trkB.t1) were found to be localized primarily to the postsynaptic acetylcholine receptor- (AChR-) rich membrane at neuromuscular junctions. In vivo, dominant-negative manipulation of TrkB signaling using adenovirus to overexpress trkB.t1 in mouse sternomastoid muscle fibers resulted in the disassembly of postsynaptic AChR clusters at neuromuscular junctions, similar to that observed in mutant trkB+/- mice. When TrkB-mediated signaling was disrupted in cultured myotubes in the absence of motor nerve terminals and Schwann cells, agrin-induced AChR clusters were also disassembled. These results demonstrate a novel role for neurotrophin signaling through TrkB receptors on muscle fibers in the ongoing maintenance of postsynaptic AChR regions.

Lambert-Eaton antibodies inhibit Ca2+ currents but paradoxically increase exocytosis during stimulus trains in bovine adrenal chromaffin cells.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease that affects neurotransmitter release at peripheral synapses. LEMS antibodies inhibit Ca2+ currents in excitable cells, but it is not known whether there are additional effects on stimulus-secretion coupling. The effect of LEMS antibodies on Ca2+ currents and exocytosis was studied in bovine adrenal chromaffin cells using whole-cell voltage clamp in perforated-patch recordings. Purified LEMS IgGs from five patients inhibited N- and P/Q-type Ca2+ current components to different extents. The reduction in Ca2+ current resulted in smaller exocytotic responses to single depolarizing pulses, but the normal relationship between integrated Ca2+ entry and exocytosis (Enisch and Nowycky, 1996) was preserved. The hallmark of LEMS is a large potentiation of neuromuscular transmission after high-frequency stimulation. In chromaffin cells, stimulus trains can induce activity-dependent enhancement of the Ca2+-exocytosis relationship. Enhancement during trains occurs most frequently when pulses are brief and evoke very small amounts of Ca2+ entry (Engisch et al., 1997). LEMS antibody treatment increased the percentage of trains eliciting enhancement through two mechanisms: (1) by reducing Ca2+ entry and (2) through a Ca2+-independent effect on the process of enhancement. This leads to a paradoxical increase in the amount of exocytosis during stimulus trains, despite inhibition of Ca2+ currents.

Interaction between the skeletal muscle type 1 Na+ channel promoter E-box and an upstream repressor element. Release of repression by myogenin.

We have defined how four elements that regulate expression of the rat skeletal muscle type 1 sodium channel (SkM1) gene cooperate to yield specific expression in differentiated muscle. A basal promoter region containing within it a promoter E-box (-31/-26) is broadly expressed in many cells, including myoblasts and myotubes; mutations within the promoter E-box that disrupt binding of the myogenic basic helix-loop-helix (bHLH) factors reduce expression in all cell types only slightly. Sequential addition of upstream elements to the wild-type promoter confer increasing specificity of expression in differentiated cells, even though all three upstream elements, including a positive element (-85/-57), a repressor E-box (-90/-85), and upstream repressor sequences (-135/-95), bind ubiquitously expressed transcription factors. Mutations in the promoter E-box that disrupt the binding of the bHLH factors counteract the specificity conferred by addition of the upstream elements, with the greatest interaction observed between the upstream repressor sequences and the promoter E-box. Forced expression of myogenin in myoblasts releases repression exerted by the upstream repressor sequences in conjunction with the wild-type, but not mutant, promoter E-box, and also initiates expression of the endogenous SkM1 protein. Our data suggest that particular myogenic bHLH proteins bound at the promoter E-box control expression of SkM1 by releasing repression exerted by upstream repressor sequences in differentiated muscle cells.

Two E-boxes are the focal point of muscle-specific skeletal muscle type 1 Na+ channel gene expression.

We have characterized a group of cis-regulatory elements that control muscle-specific expression of the rat skeletal muscle type 1 sodium channel (SkM1) gene. These elements are located within a 3. 1-kilobase fragment that encompasses the 5'-flanking region, first exon, and part of the first intron of SkM1. We sequenced the region between -1062 and +311 and determined the start sites of transcription; multiple sites were identified between +1 and +30. The basal promoter (-65/+11) lacks cell-type specificity, while an upstream repressor (-174/-65) confers muscle-specific expression. A positive element (+49/+254) increases muscle-specific expression. Within these broad elements, two E boxes play a pivotal role. One E box at -31/-26 within the promoter, acting in part through its ability to bind the myogenic basic helix-loop-helix proteins, recruits additional factor(s) that bind elsewhere within the SkM1 sequence to control positive expression of the gene. A second E box at -90/-85 within the repressor controls negative regulation of the gene and acts through a different complex of proteins. Several of these cis-regulatory elements share both sequence and functional similarities with cis-regulatory elements of the acetylcholine receptor delta-subunit; the different arrangement of these elements may contribute to unique expression patterns for the two genes.

Loss of electrical excitability in an animal model of acute quadriplegic myopathy.

In rats treated with high-dose corticosteroids, skeletal muscle that is denervated in vivo (steroid-denervated [S-D]) develops electrical inexcitability similar to that seen in patients with acute quadriplegic myopathy. In studies of affected muscles in vitro, the majority of S-D fibers failed to generate action potentials in response to intracellular stimulation although the average resting potential of these fibers was no different from that of control denervated muscle. The downregulation of membrane chloride conductance (G[Cl]) seen in normal muscle after denervation did not occur in S-D muscle. Although block of chloride channels in S-D muscle produced high specific membrane resistance, comparable to similarly treated control denervated muscle, and partially restored excitability in many fibers, action potential amplitude was still reduced in S-D fibers, suggesting a concomitant reduction in sodium current. 3H-saxitoxin binding measurements revealed a reduction in the density of the adult muscle sodium channel isoform in S-D muscle, suggesting that a decrease in the number of sodium channels present may play a role in the reduction of sodium current, although altered properties of channels may also contribute. The weakness seen in S-D muscle may involve the interaction of a number of factors that modify membrane excitability, including membrane depolarization, persistence of G(Cl), and reduced voltage-gated sodium currents.

Direct muscle stimulation in acute quadriplegic myopathy.

We have previously found that muscle is electrically inexcitable in severe acute quadriplegic myopathy (AQM). In contrast, muscle retains normal electrical excitability in peripheral neuropathy. To study the relationship between muscle electrical excitability and all types of flaccid weakness occurring in the intensive care unit, we identified 14 critically ill, weak patients and measured the amplitude of compound muscle action potentials (CMAPs) obtained with direct muscle stimulation (dmCMAP) and with nerve stimulation (neCMAP). In 11 of 14 patients dmCMAP amplitudes were reduced and the ratio of the neCMAP amplitude to the dmCMAP amplitude (nerve/muscle ratio) was indicative of loss of muscle electrical excitability. In 2 other patients, the nerve/muscle ratio indicated neuropathy. Direct muscle stimulation may allow differentiation of AQM from neuropathy even in comatose or encephalopathic critically ill patients. AQM may be more common than has previously been appreciated.

Mice lacking the myotonic dystrophy protein kinase develop a late onset progressive myopathy.

Myotonic dystrophy (DM) is an autosomal dominant disorder resulting from the expansion of a CTG repeat in the 3' untranslated region of a putative protein kinase (DMPK). To elucidate the role of DMPK in DM pathogenesis we have developed Dmpk deficient (Dmpk-/-) mice. Dmpk-/-mice develop a late-onset, progressive skeletal myopathy that shares some pathological features with DM. Muscles from mature mice show variation in fibre size, increased fibre degeneration and fibrosis. Adult Dmpk-/-mice show ultrastructural changes in muscle and a 50% decrease in force generation compared to young mice. Our results indicate that DMPK may be necessary for the maintenance of skeletal muscle structure and function and suggest that a decrease in DMPK levels may contribute to DM pathology.

Treatment of severe torsional malalignment syndrome.

Over a 7-year period (1984-1991), nine patients (aged 10-18 years) with 13 involved extremities were operatively treated for symptomatic severe torsional malalignment of the lower extremity and associated patellofemoral pathology. Physical examination and analysis of gait revealed severe rotational deformity characterized by excessive femoral anteversion and external tibial torsion. The cosmetic and functional pathologic effect of this torsional malalignment was centered about the knee joint. In all patients, conservative treatment, including therapy for muscle strengthening and nonsteroidal medication, was unsuccessful in alleviating suspected patellofemoral pain. Subsequent definitive operative treatment in all 13 extremities consisted of corrective osteotomies, internally rotating the distal part of the tibia or externally rotating the distal part of the femur or both. Osteotomies were performed as close to the knee joint as possible. No additional soft-tissue procedures were performed directly to affect patellar tracking. All osteotomies healed without complications. At an average follow-up of 2 years + 7 months (range, 18-48 months) overall, patients had an improvement in gait pattern, extremity appearance, and a marked decrease in knee pain.

Muscle is electrically inexcitable in acute quadriplegic myopathy.

We directly stimulated muscle in three patients with acute quadriplegic myopathy to determine whether paralyzed muscle in this syndrome is electrically excitable. Two of the patients had been treated with neuromuscular blocking agents and corticosteroids, and one patient had been treated with corticosteroids alone. We found that paralyzed muscle is electrically inexcitable in affected patients. Muscle regained electrical excitability over weeks to months. The recovery of muscle excitability paralleled the clinical recovery of patients, suggesting that paralysis in this syndrome is secondary to electrical inexcitability of muscle membrane.